According to their pharmacological characteristics, GPCR ligands are currently classified into agonists, neutral antagonists, and inverse agonists. Inverse agonists are drugs that can reduce GPCR-mediated G protein activation observed in the absence of ligands (basal GPCR activity). At present, little is known about the nature of the structural changes that inverse agonists induce in their target receptors. [unreadable] To shed light on this issue, we have used the rat M3 muscarinic acetylcholine receptor, a prototypic class I GPCR, as a model system. To monitor ligand-induced changes in receptor structure, we employed a recently developed in situ disulfide cross-linking strategy that allows the detection of disulfide bond formation between Cys residues that are adjacent to each other in the three-dimensional (3D) structure of the receptor. One major advantage of this strategy is that ligand-dependent conformational changes can be detected in receptors present in their native membrane environment, without the need for any receptor purification and reconstitution steps. [unreadable] We recently examined whether different classes of muscarinic ligands (full versus inverse muscarinic agonists) had different effects on the relative orientation of helix 8 relative to the C-terminus of transmembrane domain I (TM I). Helix 8 represents a cytoplasmic alpha-helical extension of TM VII to which it is connected via a short linker sequence. Considerable evidence suggests that helix 8 plays an important role in productive receptor/G protein coupling. The high-resolution structure of bovine rhodopsin indicates that several residues contained within helix 8 are located close to the cytoplasmic end of TM I. We therefore hypothesized that Cys residues substituted into this segment of TM I might serve as useful reporters to detect potential ligand-induced movements of helix 8 in disulfide cross-linking studies.[unreadable] Specifically, we introduced pairs of Cys residues into a modified version of the M3 muscarinic receptor that lacked most native Cys residues and contained two factor Xa cleavage sites within the third intracellular loop (we referred to this construct as 'M3'(3C)-Xa' receptor). We demonstrated previously that the M3'(3C)-Xa receptor exhibits ligand binding and G protein coupling properties similar to the wild-type M3 muscarinic receptor. We generated twenty double Cys mutant M3 receptors, all of which contained one Cys substitution within the cytoplasmic end of TM I (A91-N95) and a second one within the N-terminal segment of helix 8 (K548-R551). [unreadable] We demonstrated that muscarinic agonists inhibited disulfide cross-linking in the A91C/T549C and F92C/F550C double Cys mutant M3 receptors (Li et al., J. Biol. Chem. 2007 Jul 10; Epub ahead of print). In contrast, atropine and NMS enhanced disulfide bond formation in these two mutant receptors (Li et al., J. Biol. Chem. 2007 Jul 10; Epub ahead of print). Our data therefore strongly support a model in which full muscarinic agonists trigger a separation of the N-terminal segment of helix 8 from the cytoplasmic end of TM I, thus preventing the formation of disulfide cross-links between Cys residues introduced at positions 91/549 and 92/550. On the other hand, inverse muscarinic agonists are predicted to decrease the distance between the cytoplasmic end of TM I and the N-terminal portion of helix 8.[unreadable] These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most GPCRs, our findings should be of broad general relevance.